HSPA1-regulated mitophagy-necroptosis crosstalk determines the fate of brain microvascular endothelial cells after cerebral ischemia.

Ischemic stroke triggers brain microvascular endothelial dysfunction. Necroptosis (RIP3-MLKL) and PINK1-Parkin mitophagy are both implicated, but their coordination and the role of HSPA1/HSP70 remain unclear. Using oxygen-glucose deprivation (OGD) in hCMEC/D3 cells and a rat permanent middle cerebral artery occlusion (pMCAO) model, we tested whether stress-inducible HSPA1 mediates a mitochondrial "tug-of-war" between necroptosis and mitophagy. Time-course analysis identified a 4-h OGD window in which RIP3/MLKL activation and mitochondrial MLKL oligomerization peaked, while PINK1-Parkin and HSPA1 increased later. Within this window, necrostatin-1 (Nec-1) suppressed RIP3/MLKL signalling and mitochondrial MLKL oligomers, improved cell viability, and partially reshaped mitophagy markers. Rapamycin (RAPA) improved viability, upregulated PINK1, LC3-II/LC3-I and HSPA1, and reduced mitochondrial MLKL oligomers despite increased total RIP3/MLKL, consistent with enhanced autophagy and attenuated necroptotic execution. The mitochondrial HSP70 inhibitor MKT-077 reduced both MLKL oligomers and PINK1, suggesting that both pathways may be influenced by HSPA1-related activity. In pMCAO rats, Nec-1 and MKT-077, and to a lesser extent RAPA, improved neurological outcomes and reduced infarct volume; immunofluorescence further revealed increased necroptosis- and mitophagy-related signals in the peri-infarct cortex, with overlapping MLKL and PINK1 signals observed in CD31-positive endothelial cells/microvascular structures. Collectively, HSPA1 may function as a shared and limited chaperone resource that shifts from supporting necroptosis early to facilitating mitophagy as its abundance rises, thereby protecting endothelium after cerebral ischemia.